Immunoassay methods and compositions for detecting infection involving use of test antigens as cross-reactive control antigens

ABSTRACT

The present invention relates to compositions and methods involving diagnostic tests with multiple test antigens. The present invention involves the expanded use of test antigens as cross-reactive control antigens (CCAs). The invention advantageously provides for enhanced test results analysis by simultaneously providing both test antigen and CCA signal results. These results, in turn, allow useful sample comparison and cross-reference between samples to more accurately identify and verify the fidelity of test results obtained for multiple infective agents at once. The present invention may include compositions and methods for detecting infection by Zika virus or another flavivirus, and may distinguish between infections caused by genetically similar agents.

TECHNICAL FIELD

The present invention relates to compositions and methods involvingdiagnostic tests with multiple test antigens. Specifically, thisinvention relates to diagnostic compositions and methods involving theexpanded use of test antigens as cross-reactive control antigens (CCAs).The diagnostic test compositions and methods described hereinadvantageously provide for enhanced test results analysis bysimultaneously providing both test antigen and CCA signal results. Theseresults, in turn, allow useful sample comparison and cross-referencebetween samples to more accurately identify test results obtained formultiple infective agents at once. The present invention in a particularembodiment relates generally to compositions and methods for detectinginfection by an infectious agent, such as Flavivirus, including Zikavirus, and also distinguishing infections caused by genetically similaragents or presenting in subjects with similar symptomology.

BACKGROUND

Zika virus disease (Zika) is a disease caused by Zika virus that isspread to people primarily through the bite of an infected mosquito fromthe Aedes genus, mainly Aedes aegypti in tropical regions. This is thesame mosquito species that transmits dengue, chikungunya, and yellowfever. Common symptoms of Zika are fever, rash, joint pain, andconjunctivitis (red eyes). The illness is usually mild with symptomslasting for several days to a week after being bitten by an infectedmosquito.

The Zika virus was first discovered in 1947 and is named after the Zikaforest in Uganda. In 1952, the first human cases of Zika were detectedand since then, outbreaks of Zika have been reported in tropical Africa,Southeast Asia, and the Pacific Islands. Zika outbreaks have probablyoccurred in many locations but remain unrecognized because the symptomsare similar to many other diseases such as dengue and chikungunya. InMay 2015, the Pan American Health Organization (PAHO) issued an alertregarding the first confirmed Zika virus infection in Brazil and on Feb.1, 2016, the World Health Organization (WHO) declared Zika virus apublic health emergency of international concern (PHEIC). Localtransmission has been reported in many other countries and territories.

Of major concern is the effect the Zika virus may have on pregnantwomen. There is currently a very strong link between the Zika virus inpregnant women and the increase in the number of cases of babies bornwith microcephaly in Brazil and other countries, which is causing alarmworldwide. Sexual transmission of Zika virus is also of great concern,and in the United States cases of women contracting the disease fromtheir partners have been reported.

In 2004, Chikungunya virus (CHIKV) reemerged from Africa and spread tothe Indian Ocean Basin, Asia, and Europe causing explosive epidemicsaffecting millions of people. Chikungunya fever is characterized bysevere, debilitating, and often chronic arthralgia that can persist foryears, resulting in major economic as well as public health impacts.Additionally, CHIK fever is not easily diagnosed due to the overlap ininitial signs and symptoms with dengue, malaria and other acute febrileillnesses, as well as the lack of high quality, affordable,commercially-available diagnostic assays.

The majority of people infected with CHIKV become symptomatic. Theincubation period is typically 3-7 days (range, 1-12 days). The diseaseis most often characterized by acute onset of fever (typically >39° C.)and polyarthralgia. Joint symptoms can be severe and debilitating. Othersymptoms may include headache, myalgia, arthritis, conjunctivitis,nausea/vomiting, or maculopapular rash. Clinical laboratory findings caninclude lymphopenia, thrombocytopenia, elevated creatinine, and elevatedhepatic transaminases. Acute symptoms typically resolve within 7-10days. Rare complications include uveitis, retinitis, myocarditis,hepatitis, nephritis, bullous skin lesions, hemorrhage,meningoencephalitis, myelitis, Guillain-Barré syndrome, and cranialnerve palsies. Zika virus, Dengue and chikungunya viruses aretransmitted by the same mosquitoes and have similar clinical features.The viruses can circulate in the same area and can cause occasionalco-infections in the same patient. Chikungunya virus infection is morelikely to cause high fever, severe arthralgia, arthritis, rash, andlymphopenia, while dengue virus infection is more likely to causeneutropenia, thrombocytopenia, hemorrhage, shock, and death. It isimportant to differentiate viral infections because proper clinicalmanagement of can improve outcome.

Dengue, Zika, and chikungunya viruses co-circulate in the Americas.Puerto Rico has experienced eight major epidemics of these diseases inthe last 12 years. In the past year, more than 90 thousand tests havebeen run in Puerto Rico in order to screen pregnant women for recentZika infections, or diagnose Zika-suspected patients. Diagnostic testsfor dengue and chikungunya are also conducted routinely given thesimilarities of these co-endemic diseases. The arrival of Zika in theAmericas is a problem in because it generates cross reactivity withdengue in conventionally available flavivirus diagnostic tests.

Accordingly, there is a need for more efficient, improved, and moresensitive diagnostic tests that can distinguish between closely relatedinfectious agents and/or test for multiple infectious agents at once.For example, there is a strong need for diagnostic tests that providemore definitive identification of the infecting virus (e.g., Zika versusDengue). Additionally, there is need for more efficient, improved, andmore sensitive diagnostic tests that can be used to distinguish betweenvarious potential infectious agents in test subject samples where a testsubject presents with symptomology common to several potential infectiveagents and/or the test subject has been exposed to one or more potentialinfective agents.

BRIEF SUMMARY

The present invention relates to immunoassay compositions and methodsthat involve the expanded use of test antigens as cross-reactive controlantigens (CCAs) to detect and distinguish between multiple infectiveagents. Because signal results for test antigens described herein can beused to indicate not only the presence or absence of a particular targetin a test subject sample, but also illuminate the likelihood ofcross-reactivity of a test subject sample across multiple test antigens,the present invention provides a more efficient, improved, and moresensitive compositions and methods for diagnostic tests with multipletest antigens.

In one embodiment the present invention can be advantageously used tonot only detect, but to distinguish between, human antibodies to one ormore genetically similar infective agents. Thus, the present inventioncan be used to differentiate between infections caused by suchgenetically similar infective agents such as, for example, members ofthe flavivirus genus (Zika, Dengue, West Nile, etc.) or members of thealphavirus genus (chikungunya, etc.). Similar infective agents accordingto an embodiment of the present invention can be, for example,cross-reactive antigens such as the Zika E protein which is closelyrelated to the E proteins of other flavivirus.

In another embodiment, the present invention may also relate to anefficient immunoassay to detect and distinguish between human antibodiesto one or more infective, regardless of whether the infective agents aregenetically similar, giving rise to similar symptoms in a subject.

Accordingly, unlike conventionally available diagnostic tests, thepresent invention provides new in vitro tools, products, and methodsthat can be used to avoid inaccurate and confusing results stemming fromcross-reactivity between infective agents, including genetically similarinfective agents, and can aid diagnosis of infection by any one or morespecific infective agents. Several virtues of the present inventionarise from the new, expanded, and efficient use of infective targetantigens to generate both target antigen results and cross-reactivecontrol antigen results within the same assay. That is, the presentinvention is designed and deployed such that, for each sample, theresults obtained for each target antigen may not only indicate infectionfor that particular target antigen, but also may also be compared toresults obtained for a different target antigen and for a normal cellantigen to determine the likelihood of infection for any tested andcompared target antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts an ELISA-type assay wherein anti-IgM capture antibodiesare immobilized in an assay well, IgM antibodies are bound by thecapture antibodies forming antibody-antibody complexes, theantibody-antibody complex is bound to a Zika VLP forming atarget-antigen-immune complex, an anti-flavivirus antibody is bound tothe Zika VLP, and an HRP-conjugated antibody is bound to theanti-flavivirus antibody.

FIG. 1B depicts an ELISA-type assay wherein anti-IgM capture antibodiesare immobilized in an assay well, IgM antibodies are bound by thecapture antibodies forming antibody-antibody complexes, theantibody-antibody complex is cross-reactive with a CCA (e.g., DENV VLP)forming a CCA-immune complex, an anti-flavivirus antibody is bound tothe CCA, and an HRP-conjugated antibody is bound to the anti-flavivirusantibody.

FIG. 1C depicts an ELISA-type assay wherein anti-IgM capture antibodiesare immobilized in an assay well, IgM antibodies are bound by thecapture antibodies forming antibody-antibody complexes, and theantibody-antibody complex is cross-reactive with a NCA forming anNCA-immune complex. The anti-flavivirus antibody does not bind the NCA,and the HRP-conjugated antibody and the anti-flavivirus antibody will bewashed out of the assay well prior to detection.

FIG. 2 depicts an exemplary plate layout for the assay described inExample 4.

FIG. 3A and FIG. 3B depicts an exemplary Zika Interpretation Table.

FIG. 4A, FIG. 4B, and FIG. 4C depict example raw data for a panel ofZika, West Nile, Dengue, Chikungunya and normal serum samples.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, and FIG. 5E depict similar analysesto that described and depicted with respect to FIG. 4A, FIG. 4B, andFIG. 4C and can be performed using the Dengue, West Nile and Chikungunyaantigens to properly categorize these while using an appropriate CCA

DETAILED DESCRIPTION

The present disclosure provides compositions and methods for detectinginfection of a subject by an infectious agent, such as a virus.Detection methods include immunoassays that detect antibodies that binda virus envelope present in a biological sample. Antibodies specific tothe virus are determined by assaying against the target virus andagainst cross-reacting virus or viruses. A subject has been infectedwith a virus when there are more antibodies to the virus than to thecross-reacting viruses. Kits to perform the immunoassay are alsoprovided.

Definitions

The following terms used in this description are defined below.

Here, any cross-reactivity, concentration range, percentage range, ratiorange, or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. Also, any number range recited herein relating toany physical feature, such as polymer subunits, size or thickness, areto be understood to include any integer within the recited range, unlessotherwise indicated. As used herein, the term “about” means ±20% of theindicated range, value, cross-reactivity, or structure, unless otherwiseindicated.

The term “consisting essentially of” limits the scope of a claim to thespecified materials or steps, or to those materials or steps that do notmaterially affect the basic and novel characteristics of the claimedinvention. It should be understood that the terms “a” and “an” as usedherein refer to “one or more” of the enumerated components. The use ofthe alternative (e.g., “or”) should be understood to mean either one,both, or any combination thereof of the alternatives. The terms“include,” “have” and “comprise” are used synonymously, which terms andvariants thereof are intended to be construed as non-limiting.

“Target infectious agent” refers to a pathogen, the presence or absenceof which is sought to be determined by a diagnostic assay.

“Common” refers to something that is shared or in common either in wholeor in part. For example, the present invention may include common boundcomponents such as test subject sample antibodies that are used toexamine reaction with different test antigens. Also, for example,different test antigens may share common reactive or binding sites orportions with each other. Also, for example, the present invention mayinclude a common test antigen conjugate that has common reactive orbinding sites or portions with different test antigens.

“Virus-Like Particle” or “VLP” refers to a virus particles made up ofone of more viral structural proteins, but lacking the viral genome.Because VLPs lack a viral genome, they are non-infectious. In someembodiments, the VLPs are flavivirus VLPs, such as Zika virus VLPs. Insome embodiments, flavivirus VLPs include two flavivirus structuralproteins—prM/M and E.

“Cross-reactive control antigen” or “CCA” refers to one or more antigensthat are related to the target infectious agent (either by clinicalsymptom presentation or by sequence homology) and may be used fordiscerning the relative reactivity levels. In some embodiments, the CCAis a mixture of Dengue and West Nile Virus VLPs. In some embodiments,the CCA is a Zika VLP, a Dengue VLP, or a West Nile VLP. In someembodiments the CCA is a closely related protein common to otherviruses. For example, CCA closely related proteins can include the ZikaE protein which is closely related to the E proteins of otherflavivirus.

“Immunologically challenged” refers to exposure of immune cells in asubject to an infectious agent that results in a humoral immuneresponse. An immunological challenge can result from or during aninfection.

“Contacting” or “contacted” refers to placement in direct physicalassociation; includes both in solid and liquid form. “Contacting” isoften used interchangeably with “exposed.” In some cases, “contacting”refers to incubating a molecule (such as an antibody) with a biologicalsample.

“Binding agent” refers to a complementary component that has an affinityfor its binding pair. An exemplary binding agent is an antibody.

“Antibody” refers to the immunological proteins that are specificallydeveloped (either within the host body or by tissue culture methods) tohave an affinity for the target. Exemplary antibodies include IgM, IgG,and IgA isotype antibodies, and fragments thereof.

“Immune complex” refers to a protein complex that comprises an antibodybound to an antigen. In the context of the present disclosure, the term“immune complex” is used to indicate a protein complex that includes anantigen (such as a VLP) bound to at least one antibody. In some cases,the immune complex includes an antigen (such as a VLP) bound to twoseparate antigen-specific antibodies (each binding a different epitopeof the antigen or the same epitope in different regions of the VLP), orincludes an antigen (such as a VLP) bound to an antigen-specificantibody, which is further bound to a secondary antibody. The term“antibody-antigen complex” or “antibody-VLP complex” is used to refer toan antigen (or VLP) bound to one antibody. Furthermore, the term“antibody-antibody complex” is used to refer to an antibody bound to adifferent antibody (such as an antigen-specific antibody bound to asecondary antibody or a capture antibody).

“Antigen-IgM-anti-IgM complex” refers to anti-IgM antibody that pairswith an IgM antibody from the test specimen (e.g., human serum) which inturn is bound to a target antigen. The anti-IgM antibody may be bound.This creates a three-layer binding complex. While IgM antibodies arepredominantly discussed herein, the present invention may also includeother types of antibodies and proteins.

“Antigen” refers to a protein that is relevant to the target disease.The antigen may either represent the target infectious disease directlyor a cross-reactive control infectious disease. In some embodiments, anantigen is a whole virus, viral proteins, or virus like particle.

“Biological sample or sample” refers to a sample obtained from a subjectthat is tested for the presence of antibodies. Biological samplesinclude, for example, fluid, cell and/or tissue samples. In someembodiments herein, the biological sample is a fluid sample. Fluidsample include, but are not limited to, serum, blood, plasma, urine,feces, saliva, cerebral spinal fluid (CSF), bronchoalveolar lavagefluid, or amniotic fluid.

“Control” refers to a reference standard, for example a positive controlor negative control. A positive control is known to provide a positivetest result. A negative control is known to provide a negative testresult. However, the reference standard can be a theoretical or computedresult, for example a result obtained in a population.

“Normal cell antigen” or “NCA” refers to a cell expression system thatis identical to the expression system used to generate the target andcross-reactive control antigens with the exception that no antigen isexpressed. This may act as the negative control for a specimen andaccount for any background reactivity that is present within thebiological sample.

“Cross-reactive” refers to the immune response that targets homologousportions of closely related diseases (e.g., viruses) that may causefalse positive results in serological assays.

“Infectious agent” refers to a pathogen that is capable of infecting asubject. Exemplary infectious agents include Zika virus, Dengue viruses,Chikungunya virus, West Nile virus, or Yellow Fever virus.

“Cross-reacting infectious agent” refers to a disease causing agent thatis closely related (highly homologous) to the target infectious disease.In some embodiments, an antigen for a cross-reacting infectious agentcan serve as a CCA.

“Homology or homologous” refers to the degree of identity or similarityin both sequence and protein conformation between two (or more) givenprotein sequences or structures.

“Percent identity” refers to the percentage of amino acid sequenceidentity when a protein sequence is compared to a related proteinsequence. Software, such as National Center for BiotechnologyInformation BLASTP, may be used to estimate percent identity andhomology. Sequence identity/similarity refers to the identity/similaritybetween two or more nucleic acid sequences, or two or more amino acidsequences, is expressed in terms of the identity or similarity betweenthe sequences. Sequence identity can be measured in terms of percentageidentity; the higher the percentage, the more identical the sequencesare. Sequence similarity can be measured in terms of percentagesimilarity (which takes into account conservative amino acidsubstitutions); the higher the percentage, the more similar thesequences are. Homologs or orthologs of nucleic acid or amino acidsequences possess a relatively high degree of sequenceidentity/similarity when aligned using standard methods. For example,such nucleic acid or amino acid sequences can include one or moreconformational epitopes. In some embodiments, a cross-reactive antigenhas at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%sequence identity to a target antigen.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

BLASTN is used to compare nucleic acid sequences, while BLASTP is usedto compare amino acid sequences. If the two compared sequences sharehomology, then the designated output file will present those regions ofhomology as aligned sequences. If the two compared sequences do notshare homology, then the designated output file will not present alignedsequences.

“Serum” refers to the fluid portion of the blood that separates out fromclotted blood. Serum contains many proteins, including antibodies.

“Envelope protein” or “E” protein refers to a flavivirus (including Zikavirus) structural protein. The flavivirus E protein is required formembrane fusion, and is the primary antigen inducing protective immunityto flavivirus infection. Flavivirus E protein affects host range, tissuetropism and viral virulence. The flavivirus E protein contains threestructural and functional domains, DI-DIII. In mature virus particlesthe E protein forms head to tail homodimers lying flat and forming adense lattice on the viral surface.

“Premembrane (prM) protein” refers to a flavivirus structural protein.The prM protein is an approximately 25 kDa protein that is theintracellular precursor for the membrane (M) protein. prM is believed tostabilize the E protein during transport of the immature virion to thecell surface. When the virus exits the infected cell, the prM protein iscleaved to the mature M protein, which is part of the viral envelope(Reviewed in Lindenbach and Rice, In: Fields Virology, Knipe and Howley,eds., Lippincott, Williams, and Wilkins, 991-1041, 2001).

“Secondary antibody” refers to an antibody that specifically recognizesa particular isotype of antibody (for example specifically recognizesmouse IgG, human IgG or human IgM). Secondary antibodies for use withthe methods disclosed herein include, but are not limited to, anti-mouseIgG, anti-human IgG and anti-human IgM. In some embodiments, thesecondary antibody is conjugated to a detectable label, such as afluorophore, enzyme or radioisotope, to facilitate detection ofantibodies and/or immune complexes to which the secondary antibody isbound. In some embodiments the secondary antibody recognizes and Fcregion of a particular isotype of antibody.

“Subject or test subject” refers to the individual or organism fromwhich the biological sample is derived. These may include humans,non-human primates, domestic animals, including dogs, cats, horses, andcows, or reservoir species.

“Target antigen” refers to the protein(s) that represents the targetinfectious disease. These may include envelope proteins, non-structuralproteins, viral proteins or VLP.

“Control antigen” refers to the protein(s) that represent either aclosely related or symptomatically similar infectious disease. Thecontrol antigen may also represent a “normal cell antigen” where noantigen is specifically expressed but this will act as a control forbackground reactivity. These may include envelope proteins,non-structural proteins, viral proteins or VLP.

“Positive control” refers to a biological specimen that has significantaffinity for the target antigen. These may include biological samples orrecombinant specimens (for example, chimeric antibodies) that target theinfectious disease.

“Negative control” refers to a biological specimen that does not havesignificant affinity for the target antigen. This is a biological samplethat may be used to confirm that the assay was performed properly.

“Detecting reagent” or “detection reagent” refers to the material thatspecifically binds to the target, cross-reactive or control antigens.This may include a secondary antibody, an avidin complex, a DNA hybridpair or similar binding partners to specifically indicate the presenceof the antigen. The detecting reagent may also incorporate a label (forexample, enzymes such as horse radish peroxidase, fluorophores, quantumdots, etc.) that may be used for detection. In addition, separateconjugate partners that interact with the first detecting reagent may beused. For example, conjugate-HRP antibody that specifically interactswith the primary detecting reagent antibody. Specific, non-limitingexamples of labels include fluorescent tags, enzymatic linkages, andradioactive isotopes. In one example, a “labeled antibody” refers toincorporation of another molecule in the antibody. For example, thelabel is a detectable marker, such as the incorporation of aradiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by marked avidin (for example,streptavidin containing a fluorescent marker or enzymatic activity thatcan be detected by optical or colorimetric methods). Various methods oflabeling polypeptides and glycoproteins are known in the art and may beused. Examples of labels for polypeptides include, but are not limitedto, the following: radioisotopes or radionucleotides (such as 35S, 11C,13N, 15O, 18F, 19F, 99mTc, 131I, 3H, 14C, 15N, 90Y, 99Tc, 111In and125I), fluorescent labels (such as fluorescein isothiocyanate (FITC),rhodamine, lanthanide phosphors), enzymatic labels (such as horseradishperoxidase, beta-galactosidase, luciferase, alkaline phosphatase),chemiluminescent markers, biotinyl groups, predetermined polypeptideepitopes recognized by a secondary reporter (such as a leucine zipperpair sequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags), or magnetic agents, such as gadolinium chelates.In some embodiments, labels are attached by spacer arms of variouslengths to reduce potential steric hindrance.

“Immune Status Ratio or ISR” refers to the ratio of the reactivityobserved with the target antigen to the reactivity observed with theControl Antigen. That is, ISR=Target Ag OD₄₅₀÷Control Ag OD₄₅₀.

“Cut-off ISR” refers to a threshold level that is experimentallydetermined, above which a specimen is considered reactive for the targetdisease. In some embodiments, a cut-off ISR is estimated using receiveroperating characterize (ROC) analysis to optimize for sensitivity andspecificity. Equal weight may be given to both sensitivity andspecificity when performing the analysis.

“Target Ag OD₄₅₀” refers to the raw OD₄₅₀ value obtained with a sampleusing the target Antigen (e.g., Zika VLP).

“CCA OD₄₅₀” refers to the raw OD₄₅₀ value obtained with a specimen usingthe Cross-reactive Control Antigen (e.g., VLP).

“NCA OD₄₅₀” refers to the raw OD₄₅₀ value obtained with a specimen usingthe Normal Cell Antigen (NCA).

“Zika Ag/CCA (Zika ISR)” refers to the ratio of the Zika Ag OD₄₅₀ to theCCA OD₄₅₀. That is, Zika ISR=Zika Ag OD₄₅₀ CCA OD₄₅₀.

“Zika/NCA Ratio” refers to the ratio of the Zika Ag OD₄₅₀ to the NCAOD₄₅₀. That is, Zika Ag OD₄₅₀ NCA OD₄₅₀.

“CCA/NCA Ratio” refers to the ratio of the CCA OD₄₅₀ to the NCA OD₄₅₀.That is, CCA OD₄₅₀ NCA OD₄₅₀.

“ZIKA ISR” refers to the Zika Ag OD₄₅₀ to the CCA OD₄₅₀. That is, ZIKAISR=Zika Ag OD₄₅₀ CCA OD₄₅₀.

“DENV ISR” refers to the Dengue Ag OD₄₅₀ to the CCA OD₄₅₀. That is, DENVISR=DENV Ag OD₄₅₀ CCA OD₄₅₀.

The “flavivirus” genus includes, for example, the West Nile virus,dengue virus, tick-borne encephalitis virus, yellow fever virus, Zikavirus and several other viruses which may cause encephalitis, as well asinsect-specific flaviviruses (ISFs) such as cell fusing agent virus(CFAV), Palm Creek virus (PCV), and Parramatta River virus (PaRV).

The “alphavirus” genus includes, for example, alphaviruses able toinfect various vertebrates such as humans, rodents, fish, birds, andlarger mammals such as horses as well as invertebrates. Infectionscaused by members of the alphavirus genus include Chikungunya, RossRiver, Sindbis, Barmah Forest, Mayaro and O'nyong-nyong, ad Eastern,Western and Venezuelan Equine Encephalitides, etc.

Additional definitions may be set forth throughout this disclosure.

Methods and compositions for performing diagnostic assays capable ofdetecting whether a subject has been immunologically challenged by aninfectious agent are described herein.

The present invention provides for detection of a humoral immuneresponse directed against a target infectious agent (e.g., Zika virus)while reducing the incidence of false-positive results caused bycross-reactivity with non-target infectious agents that are closelyrelated to the target infectious agent. For example, differentFlaviviruses, such as dengue virus, may be cross-reactive with Zikavirus due to genetic similarities.

In some embodiments, the present invention comprises contacting anti-IgMbinding agents separately with one of a subject's test sample comprisingIgM of unknown affinity, a positive control IgM, or a negative controlIgM to form a complex of IgM bound by the anti-IgM binding agent (each,separately, a “complex” and, collectively, “complexes”). Here, a singlesubject test sample would relate to, for example, three (3) distinctcomplexes, each involving the anti-IgM binding agent and one of IgM fromthe subject's test sample, the positive control IgM, and the negativecontrol IgM. Each distinct complex is then contacted with a targetantigen derived from the target infectious agent, such as a ZikaVirus-Like Particle (VLP), or a cross-reactive control antigen (CCA),such as a Dengue VLP. If IgM are present in a test sample that bind thetarget antigen or the CCA, then the IgM will bind and capture one ormore of the antigens. In some embodiments, the complex is contacted witha normal cell antigen (NCA) control that contains no antigen that bindsthe positive control or negative control IgM. A detection reagent thatrecognizes the antigens (e.g., the Zika VLP and the CCA VLP) is thencontacted with the complex to detect whether IgM captured the targetantigen or CCA. One exemplary detection reagent is a pan-flavivirusanti-VLP antibody conjugated to horseradish peroxidase, but otherdetection reagents may also be used. The amount of bound detectionreagent is then quantified using any number of methods known in the artincluding, for example, detection of colorimetric change, fluorescence,or radiation.

The methods disclosed herein reduce the false-positive results bycomparing the amount of captured target antigen (e.g., Zika VLP) to theamount of captured CCA (e.g., dengue VLP) to generate a ratio referredto as an immune status ratio (ISR). If the ISR is above anexperimentally determined threshold, then the test sample is positivefor IgM that are specific for the target antigen; thus, indicating thatthe subject has been immunologically challenged with the targetinfectious agent. If the ISR is below an experimentally determinedthreshold, then the test sample is negative for IgM specific to thetarget antigen; thus, indicating that the subject has not beenimmunologically challenged with the target infectious agent.

In some embodiments, the method comprises using two or more targetantigens and each antigen will dually serve as a target antigen and anindividual cross-reacting antigen control.

In an example, involving a test for dengue, Zika, and chikungunya, thedengue antigen can serve as the CCA an anti-Zika IgM ELISA, the Zikaantigen can serve as the CCA for an anti-dengue IgM ELISA, and thechikungunya antigen can serve as an NCA control for both Zika and dengueELISAs while the dengue or Zika antigen will serve as the normal controlantigen for the anti-chikungunya IgM ELISA. Advantageously, thethroughput of testing of such a test is increased because the assay canbe formatted to allow simultaneous testing of multiple patients (e.g.,28 patients) for dengue, Zika, and chikungunya in a single plate.

In one embodiment, the present invention has claims directed to a methodfor detecting antibody to a target virus in a subject, comprising:contacting a biological sample obtained from a subject with anti-IgMantibody to form an IgM-anti IgM complex; contacting an antigen from thetarget virus or a cross-reactive control antigen (CCA) from a relatedvirus with the complex; incubating for a time sufficient to allowbinding of the antigen to the complex to form an antigen-IgM-anti-IgMcomplex; contacting the antigen-IgM-anti-IgM complex with a reagent thatbinds to both the target virus antigen and the CCA; and detecting thebound reagent; wherein a ratio of detected reagent for target virusantigen to detected reagent for CCA of greater than a minimum valueindicates presence of antibody to the target virus.

In one embodiment, the target virus and cross-reactive control virus areFlaviviruses. In other embodiments, the target virus and cross-reactivecontrol virus are alphaviruses.

In an aspect, the claims are directed to a method for detecting antibodyto Zika virus in a subject, comprising: contacting a biological sampleobtained from a subject with anti-IgM antibody to form an IgM-anti IgMcomplex; contacting a Zika antigen or a cross-reactive control antigen(CCA) with the complex; incubating for a time sufficient to allowbinding of the antigen to the complex to form an antigen-IgM-anti-IgMcomplex; contacting the antigen-IgM-anti-IgM complex with a reagent thatbinds to both the Zika antigen and the CCA; and detecting the boundreagent; wherein a ratio of detected reagent for Zika antigen todetected reagent for CCA greater than a minimum threshold valueindicates presence of antibody to Zika virus. In some embodiments, theCCA is either Dengue virus antigen or West Nile virus antigen or acombination of Dengue virus antigen and West Nile virus antigen. In someembodiments, the Zika antigen, Dengue virus antigen, and West Nile virusantigen is a virus-like particle (VLP).

In certain embodiments, the reagent is an antibody. In some embodiments,the antibody is labeled. In other embodiments, the ratio of greater thanabout 1.7 indicates presence of antibody to Zika virus. Generally, thebiological sample is serum, but it is not necessarily so.

In another aspect, the claims provide a method of detecting antibody toa flavivirus other than Zika virus in a subject, comprising: contactinga biological sample obtained from a subject with anti-IgM antibody toform an IgM-anti IgM complex; contacting a Zika antigen or across-reactive control antigen (CCA) or a normal cell antigen (NCA) withthe complex; incubating for a time sufficient to allow binding of theantigen to the complex to form an antigen-IgM-anti-IgM complex;contacting a reagent that binds to both the Zika antigen and the CCA;and detecting the bound reagent; wherein a ratio of detected reagent forZika antigen to detected reagent for CCA is less than about 1.5 and aratio of detected reagent for either Zika antigen or CCA to detectedreagent for NCA is greater than about 1.5 indicates presence of antibodyto flavivirus other than Zika virus.

In embodiments, the CCA is a combination of Dengue virus antigen andWest Nile virus antigen. In embodiments, the Zika antigen, Dengue virusantigen, and West Nile virus antigen is a virus-like particle (VLP).

In certain embodiments, the reagent is an antibody, and at times islabeled. In embodiments, the biological sample is serum.

The present invention also provides for a kit for detecting antibody toZika virus in a subject, comprising: a Zika antigen and a CCA; a reagentthat binds to both Zika antigen and CCA. The kit may comprise anti-IgMantibody.

In one aspect, the claims provide a composition comprising Zika antigen,and in embodiments, the Zika antigen is a VLP. In another aspect, thecomposition comprises CCA. In some embodiments, the CCA is either Denguevirus antigen or West Nile virus antigen or a mixture of Dengue virusantigen and West Nile Virus antigen. In some embodiments, the Denguevirus antigen and the West Nile Virus antigen is a VLP.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

Infectious Agents

Some infectious agents, such as viruses, are members of a genus thatcontains organisms with closely-related envelopes, coats, outermembranes, etc. As a result, antibodies in subjects infected by aparticular infectious agent may cross-react with other members of thegenus.

Without wishing to be bound by theory, cross-reactivity can be due tosimilar amino acid sequences or carbohydrate structures on proteins.Examples of infectious agents that raise cross-reacting antibodies thatmay confound diagnosis include (but are not limited to) members ofFlaviviruses, alphaviruses, Burkholderia pseudomallei, influenza A, andinfluenza B. Cross-reactivity makes it difficult to use immunoassays todiagnose infection by a particular agent. Within each genus thecross-reactivity can be significant (e.g., different flaviviruses cangenerate cross-reactive antibodies such as Zika and West Nile). Also,different alphaviruses can cause cross-reacting antibodies with oneanother, etc. But antibodies generated to a flavivirus are not expectedto be likely to cross-react with an alphavirus.

flavivirus is a genus of viruses whose members includes the West Nilevirus, dengue virus, tick-borne encephalitis virus, yellow fever virus,Zika virus, Japanese encephalitis virus and several other viruses whichmay cause encephalitis. The genus of alphaviruses also contains manypathogenic species, including Chikungunya virus, Ross River virus,Semliki Forest virus, Venezuelan equine encephalitis virus, and Westernequine encephalitis virus. Especially because Zika virus causesmicrocephaly, a birth defect where the newborn's head is smaller thannormal and is linked with serious developmental problems, diagnosis ofZika virus infection is critical. Antibodies to Zika virus cancross-react with other Flaviviruses, many of which overlap ingeographical range.

Many pathogens result in similar clinical presentation and symptoms.Accordingly, some embodiments of the instant disclosure provide methodsfor distinguishing between pathogens with similar symptoms orcross-relative structural proteins.

In the methods and compositions described herein, the target infectiousagent and one or more cross-reacting infectious agents are used asreagents. In the case of viruses, the reagents can be e.g., whole virus,typically inactivated, isolated envelope protein or proteins, orenvelope protein(s) displayed on the surface of a carrier. The envelopeprotein(s) can be isolated from virus or produced by recombinanttechniques, which are well-known. Suitable carriers include beads,yeast, bacteria, other viruses, virus-like particles, etc. Generally,the nucleotide sequence encoding the membrane protein(s) or the mostimmunogenic protein is expressed from an expression vector. Methods andsuitable expression vectors and hosts are well known.

The Zika virus, or “ZIKV,” is a flavivirus and contains a single,positive sense viral RNA of 10.7 kb in-length that translates into asingle poly-protein, which is subsequently cleaved into three structuralproteins (capsid, premembrane/membrane, envelope; C, prM/M, E) and sevennon-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5)(Kuno and Chang, Arch Virol 152, 687-696, 2007). It has been previouslydemonstrated with other Flaviviruses that expression of prM and Eglycoproteins alone can self-assemble and be secreted as immunogenicvirus-like particles (VLPs) (Chang et al., J Virol 74, 4244-4252, 2000;Davis et al., J Virol 75, 4040-4047, 2001; Chang et al., Virology 306,170-180, 2003; Konishi et al., J Virol 72, 4925-4930, 1998; Konishi etal., Vaccine 21, 3713-3720, 2003), all of which are incorporated byreference in entirety. In addition, VLPs of several non-ZIKVFlaviviruses have been previously generated (Chang et al., J Virol 74,4244-4252, 2000; Davis et al., J Virol 75, 4040-4047, 2001; Hunt et al.,J Virol Methods 97, 133-149, 2001), all of which are incorporated byreference in entirety.

An exemplary reagent is a virus-like particle (VLP) that displays theenvelope protein of a virus. Virus-like particles (VLPs) are shell-likeviruses that lack virus-specific genetic materials. The expression ofviral structural proteins can self-assemble into a VLP. VLPs may beproduced in a variety of host cells, such as in insect cells, humancells, plants, and yeast. The choice of an expression vector depends inpart upon the host cell for production. Vectors and host cells are wellknown.

Methods of producing VLPs are known in the art and described in Chang GJ et al. Virology. 2003; 306(1):170-80; Purdy DE & Chang G J. Virology.2005; 333(2):239-50; Schalich J, et al. Journal of Virology. 1996;70(7):4549-57; Allison S L, et al. Journal of Virology. 1995;69(9):5816-20; Allison S L, et al. Journal of Virology. 2003;77(21):11357-66, which are incorporated by reference herein in entirety.

In some embodiments, the target virus and CCA virus VLPs express therelevant prM and E (envelope) proteins. The prM proteins are cleaved andthe E proteins are displayed on the VLP surface. In certain embodiments,the VLPs display E proteins and are produced in mammalian cells. Thecoding sequence for prM and E are amplified from the viral genome andinserted into a plasmid vector. Expression is under control of thecytomegalovirus early gene promoter and transcriptional control elementand an engineered Japanese Encephalitis virus (JEV) signal sequenceelement is in-frame with the prM/E sequence. Other promoters and signalsequences may be used alternatively. The plasmid is transfected intomammalian cells, and the VLPs collected from cell supernatant. VLPs maybe isolated and concentrated using standard techniques and methods.

In some embodiments, the target antigen or the CCA can comprise anon-structural viral protein. For example, the target antigen cancomprise NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5, or any combinationthereof. In some embodiments, the target antigen can comprise a Cprotein. In some embodiments, the target antigen or the CCA is a VLP.The VLP can comprise E, M, C, NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5, orany combination thereof.

In some embodiments, the target and CCA virus antigens are the envelopeprotein that is produced by recombinant technology. The envelope proteincan be the precursor form or the mature, processed form. In someembodiments, the target virus antigen can be recombinant and the CCAvirus antigen can be a VLP, or vice versa. Usually the target and CCAantigens will both be the same type of antigen (e.g., VLP, recombinantenvelope, isolated protein, inactivated virus).

In some embodiments, an antigen is produced that comprises at least oneCHIKV protein. The CHIKV antigen can comprise E1, E2, or a combinationthereof. In some embodiments, the CHIKV antigen is a VLP.

Immunoassays

A variety of immunoassays are suitable for determining infection of asubject by an infectious agent, such as a Flavivirus. Immunoassays arewell known by those of ordinary skill. In a typical immunoassay thetarget antigen and control antigen (negative control) are contacted witha biological sample from a test subject. A subject can be any speciesthat is susceptible to infection by the infectious agent, and mosttypically is human. The sample is usually blood or a component of bloodthat contains antibodies, typically serum. Alternatively, the sample canbe a tissue, such as a lymph node or bone marrow. Negative and positivecontrol sera are generally included in an immunoassay. After unboundreagents are washed away, the bound antigen-antibody complex is detectedand usually quantified. A detection system can be a labeled antibody tothe antigen or to immunoglobulins in the subject's sample. Manydifferent suitable labels can be used, including radioactive labels,enzymatic labels that react with a visualizable substrate (e.g., achromogenic substrate). The assay can be performed in any medium,including in liquid or on a solid substrate.

The solid support may be any solid material known to those of ordinaryskill in the art to which the antibody may be attached. For example, thesolid support may be a test well in a microtiter plate, or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc formed of glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681.

A convenient format of an immunoassay is an ELISA. The immunoassay candetect IgM, IgG, or IgA antibodies to the target. The presence of IgMantibodies usually signals a recent infection. In an embodiment of thepresent invention, the ZIKV IgM Capture ELISA described herein is anenzyme linked capture immunoassay for the detection of human IgMantibodies targeting the ZIKV envelope glycoproteins. Polystyrenemicrotiter wells are pre-coated with capture antibodies (anti-IgM) forhuman IgM. When IgG antibodies are to be detected, then the captureantibody will be an anti-IgG (that is reactive to all sub-classes) oranti-IgG subclass antibody. It is also suitable to use a non-antibodycapture reagent that binds IgM, IgG, or IgA antibodies. For example,Staphylococcus protein A binds human IgG antibodies.

Positive control (serum that is known to have IgM antibodies to theinfectious agent Zika virus), negative control (serum that is known tonot have IgM antibodies to the infectious agent Zika virus), and unknowntest samples are diluted into a sample dilution buffer and then added tothe ELISA plate in appropriate concentrations. Following removal ofunbound serum components, VLPs of the target agent (e.g., Zika virus)and a mixture of one or more cross-reactive agents are added to separatewells. A normal cell control composition (supernatant from host cellsthat do not produce VLPs) may be added to separate wells as an internalcontrol and to assist in detecting antibodies to related, non-targetagents. After removal of unbound VLPs, the amount of bound VLPs isdetermined. The detecting reagent can be directly observable orindirectly observable. For example, the detecting reagent can be anantibody that binds a conserved sequence in the target andcross-reacting VLPs. The antibody can be labeled with a directlydetectable label, such as a radioactive label or a fluorescent label. Orthe label can be indirectly detectable, such as an enzyme that acts on achromogenic substrate that is separately added to the wells. Manydirectly detectable labels and indirectly detectable labels arewell-known.

In some embodiments, the method includes contacting a biological samplehaving antibodies with a target antigen under conditions sufficient toform a target antigen-antibody complex if antibodies that bind thetarget antigen are present in the sample; contacting the sample with across-reactive control antigen (CCA) under conditions sufficient to forma CCA-antibody complex if antibodies that cross-react or bind the CCAare present in the sample; and detecting the target antigen-antibodycomplexes and CCA-antibody complexes. In some embodiments, the methodfurther comprises contacting the sample with a normal cell antigen (NCA)under control conditions that are the same as conditions sufficient toform the target antigen-antibody complex and the CCA-antibody complexand detecting NCA-antibody complexes. The detection of NCA-antibodycomplexes serves as control for background levels of antigen-antibodycomplex formation. The target antigen-antibody complexes and theCCA-antibody complexes are collectively referred to as antigen-antibodycomplexes.

In some embodiments, the method includes contacting the sample with ananti-antibody binding agent. The anti-antibody binding agent can beimmobilized on a solid support. The anti-antibody binding agent can bein a liquid medium. In some embodiments, the anti-antibody binding agentis a capture antibody. In an alternative embodiment, the anti-antibodybinding agent is a detection antibody. The anti-antibody binding agentcan be an anti-IgM, anti-IgG, or anti-IgA antibody. In some embodiments,an anti-IgG antibody is an anti-IgG that is reactive to all sub-classesor anti-IgG subclass antibody. Exemplary anti-antibody reagentsStaphylococcus protein A binds human IgG antibodies.

In some embodiments, detecting the antigen-antibody complexes includescontacting the antigen-antibody complexes with a detection reagent thatbinds the target-antigen and the CCA. In some embodiments, the detectionreagent is an antibody. The antibody can be a pan-flavivirus antibodythat binds to more than one Flavivirus. In some embodiments, thepan-flavivirus antibody binds to Zika virus, dengue virus, West Nilevirus, yellow fever virus, Japanese encephalitis virus, or anycombination thereof. In some embodiments, the pan-flavivirus antibodybinds to Zika virus and Dengue virus. In some embodiments, thepan-flavivirus antibody binds to Zika virus, Dengue virus, and West Nilevirus. In some embodiments, the detection reagent comprises a detectablelabel. In some examples, detecting the antigen-antibody complexescomprises contacting the antigen-antibody complexes with a detectionreagent and contacting detection antibody with a secondary antibody thatbinds the detection reagent. In some embodiments, the secondary antibodycomprises a detectable label.

In certain embodiments, the secondary antibody is an anti-IgM antibodyor an anti-IgG, such as anti-human IgM antibody or an anti-human IgGantibody.

Accordingly, in some embodiments, the method includes providing acapture antibody bound to a solid support; contacting the captureantibody with the biological sample having antibodies and suspected ofhaving an infection under conditions sufficient to allow binding of thecapture antibody to antibodies present in the biological sample, therebyforming antibody-antibody complexes; contacting a portion of theantibody-antibody complexes with a target antigen under conditionssufficient for the target antigen to bind the anti-target-antigenantibodies present in the biological sample, thereby formingtarget-antigen-immune complexes; contacting a portion of theantibody-antibody complexes with a CCA under conditions sufficient forthe CCA to bind the anti-target-antigen antibodies present in thebiological sample, thereby forming CCA-immune complexes; contacting aportion of the antibody-antibody complexes with an NCA under conditionssufficient for the NCA to bind the anti-target-antigen antibodiespresent in the biological sample, thereby forming NCA-immune complexes;and detecting the presence of the immune complexes.

In some embodiments, the target-antigen is a Zika VLP and the CCA is anon-Zika flavivirus VLP, such as a Dengue VLP or a West Nile VLP. Insome embodiments, the target antigen is a Dengue VLP, and the CCA is anon-Dengue flavivirus VLP, such as a Zika VLP or a West Nile VLP. Insome embodiments, the target antigen is a West Nile VLP, and the CCA isa non-West Nile flavivirus VLP, such as a Zika VLP or a Dengue VLP. Insome embodiments, the NCA is a sample generated from cells that do notproduce a flavivirus VLP. In some embodiments, the NCA comprises acellular control that lacks expression of a VLP. In some embodiments,the NCA comprises an alphavirus VLP. In some embodiments, the NCAcomprises a CHIKV VLP.

In some embodiments, the method includes providing an anti-IgM antibodybound to a solid support; contacting the anti-IgM antibody-bound solidsupport with a biological sample having IgM antibodies and suspected ofhaving a ZIKV infection under conditions sufficient to formanti-IgM-antibody-IgM complexes; contacting a portion of theanti-IgM-antibody-IgM complexes with a ZIKV VLP under conditionssufficient for the ZIKV VLP to bind anti-ZIKV-antibodies orcross-reactive antibodies from the biological sample to formantibody-ZIKV VLP immune complexes; contacting a portion of theanti-IgM-antibody-IgM complexes with a Dengue VLP under conditionssufficient for the Dengue VLP to bind anti-Dengue-antibodies orcross-reactive antibodies from the biological sample to formantibody-Dengue VLP immune complexes; and contacting a portion of theanti-IgM-antibody-IgM complexes with a CHIK VLP under conditionssufficient for the CHIK VLP to bind anti-CHIK VLP-antibodies from thebiological sample to form antibody-CHIK VLP immune complexes; anddetecting the presence of the immune complexes.

Detecting the presence of the immune complexes may include contactingthe immune complexes with an antibody that specifically binds the VLPand comprises a detectable label.

In some embodiments, the secondary antibody is an anti-IgM antibody,such as anti-human IgM antibody. In other embodiments, the secondaryantibody is an anti-IgG antibody, such as anti-human IgG antibody.

In some embodiments of the methods of detecting ZIKV-specificantibodies, the biological sample is a biological fluid sample. In someexamples, the biological fluid sample comprises serum, blood or plasma.In particular embodiments, the biological sample comprises serum.

In certain embodiments, horse radish peroxidase (HRP) is conjugated tothe anti-VLP, e.g., antibodies to cross-reactive E protein, and theconjugate is added to each well. For example, in this embodiment, ananti-flavivirus antibody cross-reacts with West Nile, Zika, Dengue, etc.and is directly labeled to HRP. In an embodiment, HRP conjugatedantibodies can be directly used to detect differential presence of M/Ereactive antibodies, or can be done indirectly by the use of anti-mouseor other species specific antibody conjugate. In another embodiment, aprimary antibody may be unlabeled and a secondary antibody (e.g., HRPlabeled anti-mouse IgG HRP) can be used for detection.

After washing, wells are incubated with a tetramethylbenzidine (TMB)substrate. An acidic stop solution is then added and the degree ofenzymatic turnover is determined by the absorbance (optical density)measurement at 450 nanometers. If human IgM antibodies targeting theZIKV envelope glycoproteins are present, a complex is formed consistingof the IgM, antigen, and conjugate. If IgM antibodies targeting the ZIKVenvelope glycoproteins are not present, then the antigen and conjugateare washed away. A sample is deemed positive (having IgM antibodies) forthe infectious agent when the detectable signal is higher for theinfectious agent than for the cross-reacting agents.

In certain embodiments, the methods provide for distinguishing thepresence of antibodies that bind a target infectious agent fromantibodies that cross-react with cross-reactive agent. For example, themethods allow for the detection of anti-ZIKV antibodies, whiledistinguishing the positive result from cross-reaction with antibodiesthat bind, for example, Dengue virus or West Nile virus. In someembodiments, the methods allow for distinguishing between viralinfections that have similar symptoms or clinical presentation. Forexample, the methods can distinguish between one or more flavivirusesand Chikungunya virus in the same assay.

An exemplary way to calculate the relative amounts of IgM antibodies tothe target agent and the cross-reactive agent is disclosed in theExamples. Briefly, the value of the detectable signal for the targetagent is divided by the value for the cross-reactive agent. This iscalled the target ISR.

In one embodiment, for a sample to be considered as having specific IgMantibodies to the target agent, the ISR is greater than or equal to 1.1,or greater than or equal to 1.2, or greater than or equal to 1.3, orgreater than or equal to 1.4, or greater than or equal to 1.5, orgreater than or equal to 1.6, or greater than or equal to 1.7, orgreater than or equal to 1.8, or greater than or equal to 1.9, orgreater than or equal to 2.0. In this embodiment, generally, the cut-offISR is greater than or equal to 1.7 or 1.8. The cut-off value may beexperimentally determined and typically is chosen to minimize falsepositives and negatives. Similarly, for samples with target ISR valuesless than the cut-off for positive reactivity or more usually with anISR of about 1.0, a cross-reactive ISR is calculated by dividing thevalue of the signal for the cross-reactive agent by the value for thenormal cell antigen. If this ISR is greater than or equal to 1.1, orgreater than or equal to 1.2, or greater than or equal to 1.3, orgreater than or equal to 1.4, or greater than or equal to 1.5, orgreater than or equal to 1.6, or greater than or equal to 1.7, orgreater than or equal to 1.8, or greater than or equal to 1.9, orgreater than or equal to 2.0, then the sample has IgM antibodies to thecross-reactive agent(s). In this embodiment, generally, the cut-off ISRis greater than or equal to 1.7 or 1.8.

In some embodiments, the method comprises detecting and differentiatingbetween infections with different Flaviviruses (e.g., ZIKV, DENV, WNV).For example, the value of the detectable signal for the target agent isdivided by the value for the cross-reactive agent to produce the targetISR value. In some embodiments, the target agent is ZIKV and thecross-reactive agent is DENV or WNV. In some embodiments, the targetagent is DENV and the cross-reactive agent is ZIKV or WNV. In someembodiments, the target agent is WNV and the cross-reactive agent isDENV or ZIKV. A target ISR that is greater than the cut-off valueindicates that the sample comprises antibodies specific for the targetagent. The cut-off ISR may be experimentally determined and typically ischosen to minimize false positives and negatives. For example, a targetISR greater than or equal to 1.8 in that the sample is reactive for thetarget antigen. A target ISR between 1.8 and 1.6 indicates that thesample should be retested. A target ISR less than or equal to 1.6indicates that an NCA analysis should be performed. The NCA analysisincludes generating a Target antigen/NCA value by dividing the targetantigen detectable signal for the target agent by the detectable signalfor the NCA. Further, a CCA/NCA value is generated by dividing thedetectable signal for the CCA by the detectable signal for the NCA. If,for example, the Target antigen/NCA value is greater than or equal to1.7 and the CCA/NCA value is greater than or equal to 1.7, then theresults indicate that the sample is possibly reactive to the targetantigen and possibly reactive to the CCA. If, for example, the Targetantigen/NCA value is greater than or equal to 1.7 and the CCA/NCA valueis less than or equal to 1.7, then the results indicate that the sampleis reactive to the target antigen. If, for example, the Targetantigen/NCA value is less than or equal to 1.7 and the CCA/NCA value isgreater than or equal to 1.7, then the results indicate that the sampleis reactive to the CCA antigen. If, for example, the Target antigen/NCAvalue is less than or equal to 1.7 and the CCA/NCA value is less than orequal to 1.7, then the results indicate that the sample is not reactiveto the target antigen and not reactive to the CCA.

In some embodiments, the NCA sample comprises a CHIKV antigen (e.g.,VLP). If the detectable signal from the NCA-CHIKV antigen is at leastthree times higher than the detectable signal from target antigen (e.g.,ZIKV) and the CCA (e.g., DENV), then the results indicate that thesample is reactive with the NCA-CHIKV antigen.

Kits

The present disclosure provides kits for performing the assays disclosedherein. For a solid-support ELISA assay, the kit comprises the targetantigen, e.g., ZIKV antigen or VLP (Zika Ag) that comprises the Zikaenvelope glycoproteins; the Normal Cell Antigen (NCA), and optionally,the Cross-reactive Control Antigen that comprises one or morecross-reactive control antigens or VLP (CCA).

In one embodiment, all or substantially all of test components andbuffers for the different test antigens to be used as CCAs aresubstantially the same.

In some embodiments, the kit comprises a ZIKV antigen or VLP thatcomprises ZIKV envelope glycoproteins; a DENV antigen or VLP thatcomprises DENV envelope glycoproteins, and an NCA. In some embodiments,the kit comprises a ZIKV antigen or VLP that comprises ZIKV envelopeglycoproteins; a WNV antigen or VLP that comprises WNV envelopeglycoproteins, and an NCA. In some embodiments, the kit comprises a ZIKVantigen or VLP that comprises ZIKV envelope glycoproteins; a WNV antigenor VLP that comprises WNV envelope glycoproteins, a DENV antigen or VLPthat comprises DENV envelope glycoproteins, and an NCA. In someembodiments, the NCA comprises a CHIKV antigen or VLP.

Other components that may be included in the kit are: microtiter teststrips or plate that is coated with anti-IgM antibodies or the anti-IgMantibodies (not coated onto a solid support); a negative control sample,a positive control sample, a detecting agent and substrate if used withthe detecting agent, dilution buffers, wash buffers, stop solution, andinstructions.

Additional Embodiments

In one embodiment, the present invention relates to a method fordetecting antibody to a target virus in a subject, comprising: (a)contacting a biological sample obtained from a subject with anti-IgMantibody to form an IgM-anti IgM complex; (b) contacting an antigen fromthe target virus or a cross-reactive control antigen (CCA) from relatedviruses with the complex; (c) incubating for a time sufficient to allowbinding of the antigen to the complex to form an antigen-IgM-anti-IgMcomplex; (d) contacting the antigen-IgM-anti-IgM complex with a reagentthat binds to both the target virus antigen and the CCA; and (e)detecting the bound reagent; wherein a ratio of detected reagent fortarget virus antigen to detected reagent for CCA of greater than aminimum value indicates presence of antibody to the target virus. Inthis embodiment, the target virus and cross-reactive control virus(es)may be Flaviviruses. Also in this embodiment, the target virus andcross-reactive control virus(es) may be alphaviruses.

In another embodiment, the present invention comprises a method fordetecting antibody to Zika virus in a subject, comprising: (a)contacting a biological sample obtained from a subject with anti-IgMantibody to form an IgM-anti IgM complex; (b) contacting a Zika antigenor a cross-reactive control antigen (CCA) with the complex; (c)incubating for a time sufficient to allow binding of the antigen to thecomplex to form an antigen-IgM-anti-IgM complex; (d) contacting theantigen-IgM-anti-IgM complex with a reagent that binds to both the Zikaantigen and the CCA; and (e) detecting the bound reagent; wherein aratio of detected reagent for Zika antigen to detected reagent for CCAof greater than a minimum value indicates presence of antibody to Zikavirus. In this embodiment, the CCA may be either Dengue virus antigen orWest Nile virus antigen or both Dengue virus antigen and West Nile virusantigen. Also in this embodiment, the Zika antigen, Dengue virusantigen, and West Nile virus antigen may be a virus-like particle (VLP).Also in this embodiment, the reagent may be an antibody that may or maynot be labeled. Also in this embodiment, the minimum value may be 1.5,1.6, 1.7, or 1.8 or another experimentally determined ratio. Also inthis embodiment, the biological sample may be serum or plasma.

In one embodiment the present invention is a method of detectingantibody to a flavivirus other than Zika virus in a subject, comprising:(a) contacting a biological sample obtained from a subject with anti-IgMantibody to form an IgM-anti IgM complex; (b) contacting a Zika antigenor a cross-reactive control antigen (CCA) or a normal cell antigen (NCA)with the complex; (c) incubating for a time sufficient to allow bindingof the antigen to the complex to form an antigen-IgM-anti-IgM complex;(d) contacting a reagent that binds to both the Zika antigen and theCCA; and (e) detecting the bound reagent; wherein a ratio of detectedreagent for Zika antigen to detected reagent for NCA is less than anestablished value and a ratio of detected reagent for CCA to detectedreagent for NCA is greater than the established value indicates presenceof antibody to flavivirus other than Zika virus. In this embodiment, theCCA may be either Dengue virus antigen or West Nile virus antigen or acombination of Dengue virus antigen and West Nile virus antigen. Also inthis embodiment, the Zika antigen, Dengue virus antigen, and West Nilevirus antigen may be virus-like particles (VLP). Also in thisembodiment, the minimum value may be 1.5, 1.6, 1.7, or 1.8 or anotherexperimentally determined ratio. Also in this embodiment, the reagentmay be an antibody and may or may not be labeled. Also in thisembodiment, the biological sample may be serum or plasma.

In another embodiment, the present invention relates to a kit fordetecting antibody to Zika virus in a subject, comprising: (a) a Zikaantigen and a CCA; and (b) a reagent that binds to both Zika antigen andCCA. In this embodiment, the kit may further comprise an anti-IgMantibody.

In another embodiment, the present invention relates to a compositioncomprising Zika antigen that may or may not be a VLP.

In another embodiment, the present invention relates to a compositioncomprising a CCA. In this embodiment the CCA may or may not be a mixtureof Dengue virus antigen and West Nile Virus antigen. Also in thisembodiment, any Dengue virus antigen and West Nile Virus antigen may ormay not be a VLP.

In another embodiment, the present invention is a method for detectingantibody to a target infectious agent in a subject, comprising: (a)contacting a biological sample obtained from a subject with a reagentthat binds to antibody to form an antibody-reagent complex; (b)contacting an antigen from the target infectious agent or across-reactive control antigen (CCA) from a related infectious agentwith the complex; (c) incubating for a time sufficient to allow bindingof the antigen to the complex to form an antigen-antibody-reagentcomplex; (d) contacting the antigen-antibody-reagent complex with asecond reagent that binds to both the target antigen and the CCAantigen; and (e) detecting the bound second reagent; wherein a ratio ofdetected second reagent for target antigen to detected second reagentfor CCA of greater than a minimum value indicates presence of antibodyto the target infectious agent. In this embodiment, the antibody may beIgM, IgG, or IgA. In this embodiment, the infectious agent is a virus.

In one embodiment, the present invention is a diagnostic assaycomprising different test antigens, wherein each test antigen binds toeach of a common bound component and a common test antigen conjugate. Inthis embodiment, the different test antigens provide for an expanded useof test antigens as cross-reactive control antigens (CCAs). Accordingly,the diagnostic test compositions and methods described hereinadvantageously provide for test antigen and additional test antigen CCArelated signaling across, for example, different test sample wells.Here, the common bound component may be, for example, the antibody orprotein of interest from a subject sample. Here, the bound component maybe immobilized, for example, in or on a surface of a well or togetherwith or on the surface of a bead.

In an embodiment, the common test antigen conjugate may be an antibodyor a protein, or may be a mixture of one or more antibodies or of one ormore proteins, or a combination of one or more antibodies or proteins.

In an embodiment, each of the different test antigens indicates thepresence (by binding) of absence (by not binding) of a particular boundcomponent and also indicates the likelihood of cross-reactivity errorrelative to at least one other different test antigen. Each of thedifferent test antigens may separately and may simultaneously indicatethe presence of absence of a particular bound component and alsoindicate the likelihood of cross-reactivity error relative to at leastone other different test antigen.

In an embodiment, the diagnostic test assay includes different testantigens sharing a common genetic heritage or genetic similarity. Forexample, the different test antigens may be members of the same viralgenus, such as members of the flavivirus genus or the alphavirus genus.Also, the different test antigens may include components common, orshared, by or across members of the same viral genus, such as certainenvelop proteins, etc. Such commonality allows for the use of thedifferent test antigens as CCAs. In one embodiment, CCAs may be highlyhomologous or share a sequence that has at least about 70%, 75%, 80%,85%, 90%, 95%, 97%, 99%, or 100% sequence identity to a target antigen.

The present invention may also relate to a method for distinguishingbetween related infective agents in vitro comprising: at least twodifferent test antigens; and a common cross-reactive test antigenconjugate. In one embodiment, the method requires formation of a complexincluding at least one of the two different test antigens and a commonbound component. The common bound component may be, for example,antibodies or proteins in or from a test subject sample. Formation ofthe complex can allow generating a detectable signal from at least oneof the two different test antigens; and permits using each of the atleast two different test antigens as a cross-reactive control antigen.

In an embodiment of the present invention, the method comprisesestablishing or basing a quantitative threshold to determine risk ofresult bias based on cross-reactivity. This threshold is based onobservable data generated by the in vitro formation of complexesinvolving target antigen and complexes involving CCAs. This thresholdmay vary and can be determined based on observable data generated fordifferent test antigens and CCAs under study.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Preparation of ZIKV-Derived Virus-Like Particles(VLPs)

In this example, VLPs of Zika virus are prepared.

Genomic RNA was extracted from 150 μl of Vero cell culture mediuminfected with Zika virus (ZIKV) strain MR766 using a QIAamp viral RNAkit (Qiagen, Santa Clarita, Calif.). Extracted RNA was re-suspended in80 μl of diethyl pyrocarbonate-treated water (Sigma, St. Louis, Mo.) andused as a template in a reverse transcriptase-PCR (RT-PCR) for theamplification of ZIKV prM and E genes.

Amplification primers were designed based on the published ZIKV MR766sequence (GenBank: LC002520.1) with a Afell restriction enzyme siteincorporated at the 5′ terminus of the cDNA amplicon, and an in-frametermination codon followed by a NotI restriction site was introduced atthe 3′ terminus. A single cDNA fragment containing genomic nucleotides(nt) 482 to 2488 was amplified by reverse transcriptase-mediated PCR(RT-PCR) using a Titan RT-PCR Kit (Roche Molecular Biochemical,Indianapolis, Ind.). The RT-PCR product was purified using a QIAquickPCR purification kit (Qiagen), and the DNA was eluted with 50 μl of 1 mMTris-HCl (pH 7.5). The ZIKV virus cDNA amplicon was digested with AfeIIand NotI enzymes, and the resulting fragment was inserted into pcDD1vector digested with AfeII and NotI sites of to make pcDZMR. The pcDZMRplasmid contains the cytomegalovirus early gene promoter andtranscriptional control element and an engineered Japanese Encephalitisvirus (JEV) signal sequence element in frame with the ZIKV prM/Esequence. The ZIKV prM/E gene sequence was verified by bi-directionalsequencing.

For plasmid transformation into mammalian cells, COS-1 cells were grownto 75% confluence in 150-cm² culture flasks, trypsinized, andre-suspended in 4° C. phosphate-buffered saline (PBS) to a final densityof 1-2×10⁷cells/ml. Five hundred μl of cell suspension was thenelectroporated with 10 μg of plasmid DNA, using a Bio-Rad Gene Pulser IIset at 250 V and 960 μF. Cells were diluted with 25 ml of fresh mediumafter electroporation and seeded into one 75-cm2 flask. 48 hours aftertransformation, the medium was removed and replaced with fresh culturemedium containing 0.5 mg/ml G418 (Sigma). After 2-3 weeks of selection,G418-resistant colonies were cloned by limited dilution inG418-containing medium. Expression of ZIKV VLPs in the media culturesupernatants was verified using the flavivirus E protein-specific mAb4G2.

Example 2 Preparation of Cross-Reactive Control Antigen (CCA)

The Cross-reactive Control Antigen (CCA) is prepared using a mixture ofDengue and West Nile Virus-derived VLPs, generated as for the targetantigen. The relative amount of each VLP in the mixture can be optimizedby evaluating varying concentrations of Dengue and WNV VLPsindependently against samples known to have anti-Zika virus IgM,anti-Dengue virus IgM, anti-WNV virus IgM, and against normal samplesfrom uninfected subjects. Mixtures of varying ratios of WNV and DengueVLPs are also evaluated against the samples. The ratio that yields thebiggest signal spread between Dengue/WNV and Zika is chosen.

Example 3 Preparation of Normal Cell Antigen (NCA)

The Normal Cell Antigen (NCA) is prepared by collecting supernatant fromthe same cell line (COS-1) that is used to express the Zika VLP andCross-reactive Control Antigen (CCA). The NCA is diluted at anappropriate concentration in the same diluents used with the Zika VLPand CCA. The NCA provides an additional internal control to aid in theproper classification of sample status.

Example 4 IGM Capture ELISA Assay

In this Example, the procedure to perform an IgM capture ELISA with testserum from a subject is described.

Positive and negative controls should be assayed in duplicate with theZIKV Antigen (Zika Ag), Cross-reactive Control Antigen (CCA) and NormalCell Antigen (NCA) portions of assay. Unknown serum samples to be testedcan be assayed singly with the Zika Ag, CCA and NCA.

Test sera and controls are diluted to 1:100 using Sample DilutionBuffer, containing goat serum and trace non-ionic detergent in a Trisbased buffer, pH 7.4.

Anti-IgM capture antibodies are immobilized in each assay well. Seee.g., the “Anti-IgM antibody” depicted in each of FIG. 1A, FIG. 1B, andFIG. 1C. To each well is added 50 μL diluted test sera, ZIKV IgMNegative Control, and ZIKV IgM Positive Control. The top of the plate iscovered, e.g., with Parafilm® to ensure even temperature distribution inall wells from bottom and sides.

The plate is incubated at 37° C. for 1 hour in a non-gas incubator. Theanti-IgM capture antibodies bind IgM antibodies present in the test seraor in the controls (if present), respectively. After incubation, theplate is washed multiple (e.g., 6) times using approximately 300 μL or1× wash buffer made of a phosphate based buffer containing 0.1° Atween-20, pH 7.4. in each wash cycle.

Per well, 50 μL of one of Zika Ag, CCA and NCA is added. See e.g., FIG.1A (the “Zika VLP”), FIG. 1B (the “CCA”), and FIG. 1C (the “NCA”). Theplate is covered and incubated at 37° C. for 1 hour in a non-gasincubator. Following incubation, the plate is washed as described above.An exemplary plate layout for a 96-well plate is depicted in FIG. 2.

A freshly prepared conjugate solution is made by diluting theappropriate volumes of 100× enzyme conjugate (i.e., a labeled antibodysuch as, for example, an HRP labeled anti-mouse IgG or a labeled primaryantibody) into the conjugate diluent (1 part:100 parts). The enzymeconjugate comprises anti-flavivirus antibody that can bind to the ZikaAg and the CCA, but not the NCA. See e.g., FIG. 1A, FIG. 1B, and FIG.1C. The conjugate diluent contains nonfat dry milk and trace non-ionicdetergent in a tris based buffer, pH 7.4. 50 μL of conjugate solution isadded to each well. The top of the plate is covered, and the plate isincubated at 37° C. for 1 hour in a non-gas incubator. Followingincubation, the plate is washed (e.g., 6 times) using 1× wash buffer.Chromogenic substrate, such as horse radish peroxidase (HRP), that canbind to the Zika Ag, the CCA, and the NCA is added (75 μL/well). Seee.g., the “Conjugate-HRP” in each of FIG. 1A, FIG. 1B, and FIG. 1C. Theuncovered plate is incubated in the dark at room temperature (20-25° C.)for 10 minutes. After the incubation, 50 μL/well of stop solution (1NH₂SO₄) is added and the plate if further incubated for 1 minute. Afterthe incubation, the RAW OD 450 nm value is determined.

Mean ZIKV IgM Negative Control values are calculated with ZIKV Antigen,CCA, and NCA. An exemplary negative control is shown in the table belowalong with calculation of the ZIKV IgM Negative Control values.

OD₄₅₀ Zika Ag CCA NCA Replicate 1 0.108 0.103 0.095 Replicate 2 0.0920.110 0.089 Sum 0.200 0.213 0.184 Average Zika Ag = 0.200 ÷ 2 = 0.100Average CCA = 0.213 ÷ 2 = 0.107 Average NCA = 0.184 ÷ 2 = 0.092

All values are similar indicating that there are not IgM antibodies toZika or other flavivirus in the sample.

The average values are used to perform the following calculations:

Calculate the Zika Ag/CCA Ratio (Zika ISR)≡Zika Ag÷CCA:0.100÷0.107=0.935

Calculate the Zika Ag/NCA Ratio≡Zika Ag÷NCA:0.100÷0.092=1.087

Calculate the CCA/NCA Ratio≡CCA÷NCA:0.107÷0.092=1.163

The mean ZIKV IgM Positive Control values are calculated with ZIKVAntigen, CCA, and NCA. An exemplary positive control is shown in thetable below.

OD₄₅₀ Zika Ag CCA NCA Replicate 1 1.121 0.160 0.121 Replicate 2 1.2050.152 0.105 Sum 2.326 0.312 0.226 Avg 1.163 0.156 0.113 Max OD₄₅₀ =1.163 Average Zika Ag = 2.326 ÷ 2 = 1.163 Average CCA = 0.312 ÷ 2 =0.156 Average NCA = 0.226 ÷ 2 = 0.113Zika ISR (Immune Status Ratio)≡Zika Ag÷CCA ratio: 1.163+0.156=7.455MAX ISR E Max OD₄₅₀ (here the average Zika Ag)÷NCA ratio:1.163÷0.113=10.292

The ISR for Zika is within a range indicating that there is IgManti-Zika virus in the serum sample.

The table below shows values for positive and negative controls intypical assays. These values must be obtained in order to report resultsof the assay as non-fulfillment of these criteria is an indication toreagents or test procedure error requiring repetition of the assay.

Factor (For Assay Verification) Tolerance Mean Negative Control OD₄₅₀with Zika Antigen <0.300 Mean Positive Control OD₄₅₀ in ZikaAntigen >0.300 Positive Control Zika Immune Status Ratio (ZIKA ISR) >3.0Positive Control MAX Immune Status Ratio (MAX ISR) >3.0 Negative ControlZika Immune Status Ratio (ZIKA ISR) <1.7 Negative Control Zika ImmuneStatus Ratio (MAX ISR) <1.7

Example 5 Calculation and Interpretation of Results

This example teaches a method of calculating the relative amounts of IgMantibodies to the test agent and to the cross-reacting agent(s) and howto determine which agent the subject was infected with.

Properly interpreting specimen data includes the following steps: (1)determine the Zika ISR value for each specimen; (2) evaluate the ZikaISR value and determine the preliminary sample status as “Reactive forZika IgM”, “NCA Analysis Required” or “Re-Test”. See FIG. 3, the ZikaInterpretation Table. If a sample is considered “Reactive for Zika IgM”,no further analysis is required; (3) determine if a sample falls in the“Re-Test” range. If the sample is considered “Re-Test”, the specimenshould be re-run according to the instructions for use in duplicateusing Zika Ag, CCA and NCA. The Zika ISR is then re-calculated using theaverage values from the duplicate re-test run and interpreted accordingto the Zika Interpretation Table (see FIG. 3); (4) if a sample isconsidered “NCA Analysis Required”, then calculate both the Zika Ag NCARatio and the CCA NCA Ratio. NCA Analysis will also be necessary for a“Re-Test” specimen whose average Zika ISR after re-testing is <1.70; and(5) evaluate results using the Zika Interpretation Table.

As indicated above, a number of relevant figures are used in theevaluation, including the following figures.

Zika Ag OD₄₅₀: This is the raw OD₄₅₀ value obtained with a specimenusing the ZIKV Antigen (e.g., VLP).

CCA OD₄₅₀: This is the raw OD₄₅₀ value obtained with a specimen usingthe Cross-reactive Control Antigen (e.g., VLP).

NCA OD₄₅₀: This is the raw OD₄₅₀ value obtained with a specimen usingthe Normal Cell Antigen (NCA).

ZIKA ISR: This is the ratio of the Zika Ag OD₄₅₀ to the CCA OD₄₅₀. Thatis, ZIKA ISR=Zika Ag OD₄₅₀÷CCA OD₄₅₀.

ZIKA/NCA Ratio: This is the ratio of the Zika Ag OD₄₅₀ to the NCA OD₄₅₀.That is, Zika Ag OD 450÷NCA OD₄₅₀.

CCA/NCA Ratio: This is the ratio of the CCA OD₄₅₀ to the NCA OD₄₅₀. Thatis, CCA OD 450÷NCA OD₄₅₀.

MAX OD₄₅₀: This is the maximum raw OD450 value obtained for a givenspecimen from EITHER the Zika Ag OD₄₅₀ or the CCA OD₄₅₀. That is, MAXOD₄₅₀=max (Zika Ag OD₄₅₀, CCA OD₄₅₀).

MAX ISR: This is the ratio of the MAX OD₄₅₀ to the NCA OD₄₅₀. That is,MAX ISR=MAX OD₄₅₀ NCA OD₄₅₀.

First, the Zika ISR value is determined. A Table 1 setting out exemplarycriteria for interpretation of the Zika ISR value is below.

ZIKA ISR Result Interpretation ZIKA ISR ≤1.60 Non-Reactive No detectableIgM antibody, individual does for Zika IgM not appear to have recentinfection with antibodies ZIKV. To evaluate whether there has beeninfection with other Flaviviruses, determine the MAX ISR value. Anadditional sample may be tested within 7-14 days if early infection issuspected. 1.6 < ZIKA ISR < Re-test Sample may be re-assayed induplicate to 1.8 confirm the final status. ZIKA ISR ≥1.80 ReactivePresence of detectable IgM antibody, for Zika IgM possible recentinfection with ZIKV.. antibodies

If a sample falls in the “Re-test” range, this sample may be re-tested,preferably in duplicate, following the procedure disclosed herein. Theaverage ISR value from these duplicate samples is then be interpreted asnon-reactive if the ISR is <1.7 and reactive if the ISR is ≥1.7.

Overall, the following table may be used for interpretation of results.

Scenario Result Interpretation ZIKA ISR <1.70 Non-Reactive No detectableIgM antibody, AND for Zika IgM individual does not appear to MAX ISR<1.70 antibodies have recent infection with ZIKV. An additional samplemay be tested within 7-14 days if early infection is suspected. ZIKA ISR<1.70 Reactive for Presence of detectable IgM antibody AND otherpossible targeting a flavivirus may be present. MAX ISR ≥1.70 flavivirusinfection ZIKA ISR ≥1.70 Reactive Presence of detectable IgM antibody,for Zika IgM possible recent infection with ZIKV. antibodies

For additional clarity, provided below are four example specimens withsample data for evaluation:

Zika Ag CCA NCA OD450 OD450 OD450 Sample #1 1.379 0.085 0.062 Sample #20.120 0.946 0.049 Sample #3 0.131 0.416 0.102 Sample #4 0.114 0.0990.108Step 1: Determine the Specimen's Zika ISR

Zika ISR=Zika Ag OD450÷CCA OD450. The four example specimens would thenhave the following Zika ISR values:

Zika ISR Sample 16.22 Sample 0.13 Sample 0.31 Sample 1.15Step 2: Evaluate the Specimen's Zika ISR

We first look at ISR Analysis columns of the Zika Interpretation Tableto evaluate the initial result. The four example specimens would thenhave the following preliminary interpretations:

Preliminary Interpretation Sample #1 Reactive for Zika IgM Sample #2 NCAAnalysis Required Sample #3 NCA Analysis Required Sample #4 NCA AnalysisRequiredStep 3: Determine if a Sample Falls in the “Re-Test” Range

Reviewing the Zika Interpretation Table indicates that none of theseexample specimen values fall in the “re-test” range. Therefore, noduplicate testing would be required for any of these specimens.

Duplicate testing required? Sample #1 No Sample #2 No Sample #3 NoSample #4 NoStep 4: If the Sample is Considered “NCA Analysis Required”, thenCalculate Both the Zika NCA Ratio and the CCA÷NCA Ratio

The four example specimens would then have the following ratios:

Zika ÷ NCA Ratio CCA ÷ NCA Ratio Sample #1 No NCA Analysis Required NoNCA Analysis Required (Reactive for Zika IgM) (Reactive for Zika IgM)Sample #2 2.45 (0.120 ÷ 0.049) 19.31 (0.946 ÷ 0.049)  Sample #3 1.28(0.131 ÷ 0.102) 4.08 (0.416 ÷ 0.102) Sample #4 1.06 (0.114 ÷ 0.108) 0.92(0.099 ÷ 0.108)Step 5: Evaluate the Results Using the Interpretation Table

Evaluate each ratio using the Zika Interpretation Table. The fourexample specimens would then have the following final interpretations:

Interpretation Sample #1 Presumptive Zika Positive Sample #2 PossibleZika Positive Sample #3 Presumptive Other flavivirus Positive Sample #4Negative

Example 6 IGM Capture ELISA Assay Results on Clinical Samples

Clinical Specificity and Cross-Reactivity: The ZIKV IgM ELISA kit asdescribed herein was tested against 217 samples, including 89 normalhuman serum, 89 specimens that were infected with other, non-flavivirusdiseases and 32 flavivirus IgM positive specimens. An additional 7yellow fever vaccine recipients were included for this study. Of the 89normal human samples (negative), none were reactive for Zika orcross-reactive antigens. Additionally, none of the “other disease”(non-Flavivirus) specimens was reactive in the ELISA kit. Of the 32 IgMflavivirus specimens (non-Zika), 31 were correctly categorized as “Otherflavivirus possible . . . ”. None of the 32 IgM flavivirus specimens wasconsidered Zika Reactive in the ELISA kit. One of the flavivirusspecimens tested as “Non-Reactive” in the ZIKV Detect IgM ELISA kit. Ofthe 7 yellow fever vaccine recipients, 2 were classified as “Otherflavivirus possible . . . ”.

The negative percent agreements for each category of specimen are shownbelow. The positive percent agreement for correctly classifying IgMpositive flavivirus specimens as “Other flavivirus possible . . . ” isalso indicated below. This is separate from the positive percentagreement that is established for Zika IgM positive specimens(considered “Zika Reactive”).

The potential cross-reactivity with other diseases was evaluated bytesting specimens from patients with confirmed IgM antibodies to othermicroorganisms which could potentially cause false positive results.This list is composed of organisms whose infection produces symptomssimilar to those observed at the onset of Zika virus infection and alsoviral strains which have a significant likelihood to result incross-reactivity due to genetic similarity with Zika virus. Alsoincluded were organisms/strains which are likely to be observed in thecurrently affected and endemic areas (i.e., Brazil and South America)since these organisms/strains will be an important part of thedifferential diagnosis of Zika virus infection.

Sample Status Other Yellow Disease Fever Negative (non- FlavivirusVaccine (normal) Flavivirus) (non-Zika) Recipients Total Non- 89 89 1 5184 Reactive Other 0 0 31 2 33 Flavivirus Zika 0 0 0 0 0 Reactive Total89 89 32 7 217 Negative Percent Agreement 100% (89/89, 95.9%-100%)(normal): Negative Percent Agreement (Other 100% (89/89, 95.9%-100%)Disease): Negative Percent Agreement (for 100% (32/32, 89.3%-100%)Flavivirus, non-Zika)*: Negative Percent Agreement for 100% (7/7,64.6%-100%) Yellow Fever Vaccine Recipients (considered “ZikaNon-Reactive):** Total Negative Percent Agreement: 100% (217/217,98.3%-100%) Positive Percent Agreement (for 96.9% (31/32, 84.3%-99.4%)Flavivirus, non-Zika)^(τ): *These flavivirus positive specimens screenedas non-reactive for Zika. **Note: while none of the YFV recipients wereconsidered Zika Reactive, 2 of the 7 were considered “Other PossibleFlavivirus . . . ” ^(τ)These flavivirus positive specimens screened as“Other Possible Flavivirus . . . ”

% False Disease/ # Other # % False “Other Infectious agent # of # ZikaFlavivirus Non- “Zika Flavivirus Positive Sera samples Reactive PossibleReactive Reactive” Possible” Other disease Anti-Chikungunya 8 0 0 8 0%0% present (IgM virus Positive) Anti-Cytomegalovirus 10 0 0 10 0% 0%(CMV) Anti-Epstein Barr 15 0 0 15 0% 0% Virus (EBV) -CA Anti-ParvovirusB19 5 0 0 5 0% 0% Anti-Varicella 10 0 0 10 0% 0% zoster virusAnti-nuclear 10 0 0 10 0% 0% Antibodies (ANA) Rheumatoid Factor 11 0 011 0% 0% HAMA (human anti- 3 0 0 3 0% 0% mouse antibody)Anti-Malaria/anti- 7 0 0 7 0% 0% plasmodium falciparum Anti-Hepatitis(C) 10 0 0 10 0% 0% virus Flavivirus Anti-Dengue virus 14 0 14 0 0% 0%Specimens Anti-West Nile Virus 13 0 13 0 0% 0% (non-Zika, Anti-Japanese1 0 1 0 0% 0% IgM Positive) Encephalitis Anti-Saint Louis 4 0 3 1 0% 0%encephalitis (SLE) Immunization Yellow fever virus 7 0 2 5 0% N/A* toFlavivirus post-immunization *Individuals immunized against a flavivirusmay be considered false negative or false positive with a reaction thatclassifies the specimen as “other flavivirus possible . . .”

Thirteen archived Zika IgM confirmed samples (confirmed via CDC MACELISA and/or PCR) were tested using IgM Capture ELISA as describedherein. All 13 samples were reactive for Zika virus. This estimates aPositive Percent Agreement of 100% (13/13, 77.2%-100%) for thesesamples. [All confidence intervals presented are 95% confidenceintervals using the Wilson Score method.]

In a field study in Puerto Rico 44 samples representing a mixture ofnormal, confirmed Zika IgM positive and confirmed Dengue IgM positivespecimens (PCR positive for Dengue) were tested. Additionally, pairedserum samples collected from 21 individuals (42 total samples),collected during acute and convalescent time points, were tested. Allacute samples were confirmed Zika positive by PCR at CDC. The meannumber of days post onset of symptoms was ˜2.2 days (range 1-5 days).The mean number of days post onset of symptoms for the convalescentsample was ˜15.9 days (range 6-80 days). The IgM Capture ELISA detectedall 21 individuals at the convalescent time point. Additionally, threeindividuals were considered reactive with the IgM Capture ELISA at theacute phase (days 1, 4 and five post onset of symptoms). None of thesamples had values warranting a re-test.

The above data are combined into the table below. The FinalInterpretation is determined by the following method: if a sample is PCRpositive (for Zika or Dengue), then this is considered final. Otherwise,the CDC Zika MAC-ELISA result is subsequently considered as the finaldiagnosis for these analysis purposes.

Final Interpretation Positive Equivocal Negative Dengue Total IgMReactive 38 2  0 0 40 Capture Other  1^(a) 0  3^(b)  9^(c) 13 ELISAFlavivirus Non-  0 0 12 0 12 Reactive Total: 39 2 15 9 65 ^(a)Thissample was also positive for Dengue IgM antibodies, and cross-reactivityshould be considered. This was classified as Zika positive based solelyon the CDC MAC-ELISA results. ^(b)All 3 “Other Flavivirus” reactionswere Positive for Dengue IgM antibodies with an IgM Capture ELISA todetect anti-Dengue virus IgM antibodies. ^(c)These 9 specimens wereconfirmed Dengue positive by PCR and classified as such.

Combining all data (from 13 confirmed Zika specimens) (n=13) and thespecificity and cross-reactivity data (n=217) with the testing at PuertoRico (n=65), the following overall performance characteristics for 295total samples by IgM Capture ELISA are summarized in the table below.

Sample Status Other Yellow InBios ZIKV Disease Fever Detect ™ Zika (non-Flavivirus Vaccine Negative IgM ELISA Positive Equivocal Flavivirus)(non-Zika) Recipients (normal) Total Zika Reactive 51 2 0 0 0  0 53Other  1^(a) 0 0 40 2   3^(b) 46 Flavivirus Non-Reactive  0 0 89 1 5 101196 Total 52 2 89 41 7 104 295 ^(a)This sample was also positive forDengue IgM antibodies and cross-reactivity should be considered. Thiswas classified as Zika positive based solely on the CDC MAC-ELISAresults ^(b)All 3 of these “Other Flavivirus Possible” reactions werePositive for Dengue IgM antibodies with the DENV Detect IgM CaptureELISA.

Positive Percent Agreement 98.1% (51/52, 89.9%-99.7%)^(a) (forpresumptive Zika Positive): Positive Percent Agreement 97.6% (40/41,87.4%-99.6%) (for Flavivirus, non-Zika)^(b): Negative Percent Agreement97.1% (101/104, 91.9%-99.0%)^(c) (normal): Negative Percent Agreement100% (89/89, 95.9%-100%) (Other Disease): Negative Percent Agreement100% (41/41, 91.4%-100%) (for Flavivirus, non-Zika)^(d): NegativePercent Agreement 100% (7/7, 64.6%-100%) (for Yellow Fever VaccineRecipients, considered “Zika Non- Reactive”)^(e): Total Negative PercentAgreement: 98.8% (238/241, 96.4%-99.6%) ^(a)The one specimen missed heretested as “Other Flavivirus Possible” and was also positive for DengueIgM antibodies. ^(b)These flavivirus positive specimens screened as“Other Flavivirus Possible . . . ” ^(c)All 3 of these false positivespecimens were classified as “Other Flavivirus Possible” and werePositive for Dengue IgM antibodies with the DENV Detect IgM CaptureELISA. ^(d)These are the flavivirus positive specimens that screened asnon-reactive for Zika. eNote: while none of the YFV recipients wereconsidered Zika Reactive, 2 of the 7 were considered “Other FlavivirusPossible . . . ”

Interference testing: Potentially interfering substances commonlyoccurring in serum were evaluated with the IgM Capture ELISA.Interfering substances included bilirubin (0.2 mg/mL), hemoglobin (160mg/mL), albumin (150 mg/mL) and cholesterol (5 mg/mL). These interferingsubstances were spiked into low reactive (n=3) and normal human serumsamples (n=3) to evaluate their impact on assay performance. None of theinterfering substances caused a statistically significant change in theISR value for either the low reactive samples or normal human serumsamples evaluated and did not alter the interpretation results.

Additionally, 3 human anti-mouse antibody (HAMA) serum samples and 3rheumatoid factor (RF) positive serum samples were acquired fromcommercial vendors. Each HAMA and RF sample was mixed with a Zika lowreactive specimen (ISR ˜2.5) in the kit sample dilution buffer(equivalent to a 1:1 dilution of the Zika sample:Interfering sample) andthe assay was performed as described herein. Of the 3 HAMA samples, nonealtered the reactivity of the low positive. Of the 3 RF samples, 2 outof the 3 (66.6%) diminished the ISR value substantially enough to alterthe sample from a “Reactive” status to a “Non-Reactive” status.

Effect on Low Interfering Concentration Reactive Effect on NegativeSubstance Tested Specimens Specimens Bilirubin  0.2 mg/mL None Noneobserved (0/3) observed (0/3) Hemoglobin 160 mg/mL None None observed(0/3) observed (0/3) Albumin 150 mg/mL None None observed (0/3) observed(0/3) Cholesterol  5 mg/mL None None observed (0/3) observed (0/3) HAMAVaries None None observed (see observed cross-reactivity table) (0/3) RFVaries Lowered None observed (see reactivity cross-reactivity table)(2/3)

Example 7

The IgM ELISA as described herein was tested with serum samples known tobe positive for ZIKV, DENV, WNV, or CHIKV.

The ZIKV positive serum samples were obtained from CDC panels that wereconfirmed by CDC MAC ELISA and PRNT. The WNV positive serum samples weresourced from a reference lab in South Dakota during a WNV outbreak. TheDENV positive serum samples were sourced from a reference lab in Utahwith confirmed Dengue infections. The CHIKV positive serum samples weresourced from a commercial vendor. 96-well ELISA plates were sourced fromInBios International Inc. (Part #500613) and came pre-coated withanti-Human IgM antibody.

Serum specimens were diluted into a sample dilution buffer (InBiosInternational Part #500241D) at a 1:100 dilution and added to theappropriate wells (50 μL per well). Each plate was covered and incubatedat 37° C. for 1 hour.

After incubation, plates were washed 6 times with an automated ELISAplate washer (BioTek ELX405) using a phosphate buffered saline (PBS)buffer containing 0.05% tween-20.

Antigens (50 μL per well) were then directly added to the appropriatewells such that each serum specimen is incubated with ZIKV antigen (ZikaVLP), DENV antigen (Dengue VLP), WNV antigen (WN VLP), CHIKV antigen(Chik VLP), CCA and NCA. The ELISA plates were covered and incubated at37° C. for 1 hour.

After incubation, the ELISA plates were washed 6 times using anautomated automated ELISA plate washer (BioTek ELX405) using a phosphatebuffered saline (PBS) buffer containing 0.05% tween-20.

A cocktail of pan-flavivirus monoclonal antibodies (targeting flavivirusenvelope protein) and a CHIKV antibody (targeting the Chikungunyaenvelope protein) were added to all of the wells. The ELISA plates werecovered and incubated at 37° C. for 30 minutes.

After incubation, the ELISA plates were washed 6 times using anautomated ELISA plate washer (BioTek ELX405) using a phosphate bufferedsaline (PBS) buffer containing 0.05% tween-20.

A conjugate solution (anti-mouse IgG-HRP) was then added to each well(50 μL per well). The ELISA plates were covered and incubated at 37° C.for 30 minutes.

After incubation, the ELISA plates were washed 6 times using anautomated ELISA plate washer (BioTek ELX405) using a phosphate bufferedsaline (PBS) buffer containing 0.05% tween-20.

After washing, a TMB substrate was added to each well (75 μL per well)causing a color change proportional to the amount of HRP (horse-radishperoxidase) present in each complex.

The reaction was stopped with the addition of 50 μL of 1N H₂SO₄ and theraw optical density (OD) was measured at 450 nm using an ELISA platereader.

Analysis:

The initial analysis was performed using the Zika antigen, the CCAantigen and NCA control. The CCA was comprised of a combination ofDengue antigen and West Nile antigen. An algorithm that aptly appliesfor discriminating between Zika, other Flaviviruses and Negativespecimens can be demonstrated as such:

(1) If the raw Zika OD₄₅₀ is ≥3.0×the Negative Control OD₄₅₀ AND theZika ISR≥1.70, then the specimen is considered Presumptive Zika Positive

(2) OTHERWISE, if the CCA/NCA ratio ≥3.0, then the specimen isconsidered Presumptive Other Flavivirus Positive

(3) OTHERWISE, the sample is considered Negative

Example raw data for a panel of Zika, West Nile, Dengue, Chikungunya andnormal serum samples are shown in FIG. 4A, FIG. 4B, and FIG. 5B. Theassay was performed simultaneously with all of the specimens using thesame buffers for all steps except for the target antigen. An exampleanalysis using the algorithm described above is shown using data fromthe Zika antigen, CCA and NCA. This analysis accurately categorizes theZika positive, Other Flavivirus and Negative serum specimens. Asdemonstrated in FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, and FIG. 5E, similaranalyses can be readily performed using the Dengue, West Nile andChikungunya antigens to properly categorize these while using anappropriate CCA.

From the foregoing it will be appreciated that, although specificembodiments have been described herein for purposes of illustration,various modifications may be made without deviating from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

The invention claimed is:
 1. A diagnostic assay comprising a commonbound component, a common test antigen conjugate, and two or moredifferent test antigens, wherein: i. each test antigen binds to each ofthe common bound component and the common test antigen conjugate; ii.each of the different test antigens indicates the presence or absence ofa particular bound component; iii. the different test antigens arederived from different species within a shared viral genus, wherein theshared viral genus is selected from the flavivirus genus and thealphavirus genus; iv. at least one of the different test antigens is across-reactive control antigen; and v. the ratio of binding of each ofthe different test antigens to the common bound component indicates thelikelihood of cross-reactivity error.
 2. The diagnostic assay of claim1, wherein the different test antigens are derived from two or moreviruses selected from Zika virus, dengue virus, and West Nile virus, orderivations thereof.
 3. The diagnostic assay of claim 1, wherein thedifferent test antigens are derived from two of more viruses selectedfrom Chikungunya virus, Ross River virus, Semliki Forest virus,Venezuelan equine encephalitis virus, Eastern equine encephalitis virus,and Western equine encephalitis virus, or derivations thereof.
 4. Thediagnostic assay of claim 1, wherein each of the different test antigensis a cross-reactive control.
 5. The diagnostic assay of claim 1, whereinthe different test antigens are analyzed substantially simultaneously.6. The diagnostic assay of claim 1, wherein at least one different testantigen is a virus-like particle.
 7. The diagnostic assay of claim 1,wherein the common test antigen conjugate also provides a detectablesignal.
 8. The diagnostic assay of claim 7, wherein a material used forthe detectable signal is the same for the different test antigens. 9.The diagnostic assay of claim 7, wherein the presence or absence of eachdifferent test antigen is indicated by relative signal strength.
 10. Thediagnostic assay of claim 1, wherein the particular bound component isan antibody.
 11. The diagnostic assay of claim 1, wherein the particularbound component is derived from a test subject sample.
 12. A kitcomprising the diagnostic assay of claim
 1. 13. A method fordistinguishing between related infective agents in vitro, comprising:generating a detectable signal from at least one of at least twodifferent test antigens; and using each of the at least two differenttest antigens as a cross-reactive control antigen; wherein i. each testantigen binds to each of a common bound component and a common testantigen conjugate; ii. each of the different test antigens indicates thepresence or absence of a particular bound component; and iii. thedifferent test antigens are derived from different species within ashared viral genus, wherein the shared viral genus is selected from theflavivirus genus and the alphavirus genus.
 14. The method of claim 13,further comprising: establishing a threshold based on detectable signalstrength; and distinguishing between the related infective agents basedon relative signal strength.
 15. The method of claim 13, furthercomprising including a test antigen to an unrelated infective agent,wherein the unrelated infective agent produces similar symptoms in atest subject or is an infective agent to which the test subject may havebeen exposed.
 16. The method of claim 13, wherein the related infectiveagents are selected from the Zika virus, dengue virus, and West Nilevirus, or derivations thereof.
 17. The method of claim 13, wherein therelated infective agents are selected from the Chikungunya virus, RossRiver virus, Semliki Forest virus, Venezuelan equine encephalitis virus,Eastern equine encephalitis virus, and Western equine encephalitisvirus, or derivations thereof.
 18. The method of claim 15, wherein theunrelated infective agent is chikungunya.
 19. The diagnostic assay ofclaim 1, wherein the common bound component comprises IgM antibodies.20. The diagnostic assay of claim 1, wherein the particular boundcomponent is an IgM antibody that binds at least one test antigen. 21.The kit of claim 12, wherein the kit comprises anti-IgM antibodies,normal cell antigen, a negative control sample, a positive controlsample, a detecting agent and buffers, and wherein the anti-IgM agent,and buffers for use with each of the different test antigens are thesame.